Interleukin-33 (IL-33), also called IL-1F11, is a member of the IL-1 family of cytokines that stimulates the generation of cells, cytokines, and immunoglobulins characteristic of a type two immune response. IL-33 is a 270 amino acid protein, consisting of two domains: a homeodomain and a cytokine (IL-1-like) domain. The homeodomain contains a nuclear localization signal (NLS). IL-33 mediates signal transduction through ST2, a receptor expressed on Th2 cells, mast cells and a wide variety of other cell types.
Schmitz et al. first identified IL-33 as the ligand for the orphan receptor ST2 (also called IL-1R4) (Schmitz et al., Immunity 23(5)479-90 (2005)). IL-33 receptor is formed from heterodimeric molecules. ST2 and IL-1R accessory protein (IL-1RAcP) dimerize to form an IL-33 receptor (ST2:IL-1RAcP). IL-1RAcP is a shared component of receptors for IL-1α, IL-1β, IL-1F6, IL1F8, and IL1F9. IL-1RAcP is not required for binding, but is critical for signaling. The TIR-domain of the IL-33 receptor recruits MyD88 and TRAF6, and the receptor signal results in activation of NFκB and MAP Kinase pathways (Oboki et al., Allergology International 59:143-160 (2010)). IL-33 receptor may potentially associate with other receptors and has been reported to interact with c-kit on mast cells (Drube et al., Blood 115:3899-3906 (2010))
Recently, IL-33 has been shown to bind a second IL-33 receptor heterodimeric complex: ST2 also forms a complex with another IL-1R family molecule, “single Ig IL-1R-related molecule” (SIGIRR) (also called Toll IL-1R8 (TIR8)) to form ST2:SIGIRR. SIGIRR/TIR8 is considered to act as a negative regulator of IL-1R and Toll-like receptor (TLR)-mediated immune responses (Garlanda et al., Trends Immunol. 30:439-46 (2009)). In contrast to ST2:IL-1RAcP, ST2:SIGIRR seems to act as a negative regulator of IL-33 (Oboki et al. (2010)).
The only known ligand of the ST2 receptor is IL-33 (see, e.g., Schmitz et al., Immunity 23(5)479-90 (2005); Chackerian et al., J. Immunol. 179(4):2551-5 (2007)). The ST2 receptor is expressed at baseline by Th2 cells and mast cells, both cell types are known to be important mediators of allergic asthma. The extracellular form of IL-33 stimulates target cells by binding to ST2 and subsequently activates NFκB and MAP Kinase pathways, leading to a range of functional responses including production of cytokines and chemokines. Soluble ST2 (sST2) is thought to be a decoy receptor, preventing IL-33 signaling.
In humans, IL-33 was found to be expressed constitutively in smooth muscle and in bronchial epithelia. Expression can be induced by IL-1β and TNF-α in lung and dermal fibroblasts (Schmitz et al. (2005)). The levels of soluble ST2 protein and IL-33 mRNA/protein are increased in sera and tissues from patients with asthma (Oboki et al., Allergology International 59:143-160 (2010)).
In vivo, IL-33 induces the expression of IL-4, IL-5, and IL-13 and leads to severe pathological changes in mucosal organs. Administration of IL-33 to mice has potent inflammatory effects, including massive blood eosinophilia, increased IL-5 and IgE serum levels, and goblet cell hyperplasia at mucosal surfaces (Schmitz et al. (2005)). Intraperitoneal or intranasal administration of IL-33 to mice led to induction of eosinophilic inflammation in the pulmonary and intestinal mucosa through the IL-13 and STAT6-dependent pathways (Oboki et al. (2010)). Accordingly, IL-33 may play a role in allergic diseases such as asthma and other inflammatory airway diseases.
Some reports in the literature suggest that goblet cells secrete CXCL8/IL-8, and this is increased by IL-33 through ST2R-ERK pathway, suggesting a mechanism for enhanced airway inflammation in the asthmatic airway with goblet cell metaplasia (Clin Exp Allergy. 2014 April; 44(4):540-52)
Therefore there has been interest in IL-33 as a therapeutic target. However, to date the therapeutic benefits of blocking this therapeutic target have yet to be fully realised. The present inventors have established for the first time that IL-33 exists in a reduced form (also referred to herein as redIL-33) and an oxidized form. The inventors have characterized the oxidized form of IL-33 for the first time as described herein. In vitro and in vivo studies by the inventors have shown that disappearance of redIL-33 (the reduced form) correlated with the appearance of oxidised IL-33. In physiological fluids in vitro, redIL-33 is rapidly oxidised to form a disulphide bonded form. The oxidized form (also referred to herein as IL-33-DSB) has at least one (e.g. two) disulfide bonds, in between the cysteines selected from the group Cys208, Cys 227, Cys 232 and Cys259, (numbered with reference to full length human IL-33 as disclosed in UniProt O97560, residues 112 to 270 of which are recited within SEQ ID NO. 632). It has previously been unappreciated that the commercially available assays seem to predominantly detect this oxidized form. The present inventors therefore needed to devise assays to selectively detect the redIL-33 that is responsible for interacting with ST2 and driving biological activity associated with IL-33 release.
The reduced form appears to be the active form of the protein which generates the signal cascade and in fact in vivo it appears that the reduced form is converted into the oxidized form as a mechanism for terminating the signaling through ST2. The present inventors have found that redIL-33 binds ST2 (FIG. 24A). In contrast, IL-33-DSB showed no ST2 binding (FIG. 24B). This has lead the inventors to hypothesize that in vivo oxidation could be a mechanism to turn off IL-33-ST2 activity. In addition, the present inventors have established for the first time that the oxidized form of IL-33 (IL-33-DSB) binds to receptor for advanced glycation end products (RAGE) and signals through this alternative pathway (FIG. 56).
The present inventors believe that this understanding may be utilized to generate more effective therapeutic agents. In one embodiment, the present inventors have identified an antibody that preferentially binds the oxidized form but surprisingly attenuates the activity of the reduced form by essentially catalyzing the conversion of the reduced form to the oxidized form. Advantageously this mechanism simply augments the in vivo mechanism for terminating the signaling through ST2.
In another embodiment, the inventors have identified an antibody that preferentially binds the reduced form of IL-33 (redIL-33), with femtomolar affinity, and attenuates and/or inhibits IL-33 mediated signaling through ST2. This antibody provides for the first time a mechanism for treating or preventing IL-33/ST2 mediated inflammatory responses.
In a yet further embodiment, the antibodies of the present invention can attenuate or inhibit the previously unrecognized signaling pathway for IL-33-DSB through RAGE, and any IL-33/RAGE mediated effects. The antibodies of the present invention may act by directly binding of IL-33-DSB and attenuating or inhibiting the ligand/receptor interaction with RAGE or alternatively may bind to redIL-33 and prevent or reduce its conversion to the IL-33-DSB thereby indirectly attenuating or inhibiting the ligand/receptor interaction with RAGE.